Tuberculosis (TB) is the leading cause of death due to a single bacterial pathogen. The AIDS epidemic has impacted the resurgence of TB, as HIV-infected persons have a 10% annual risk of reactivation vs. an -10% lifetime risk for immunocompetent persons. Since one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb) and at risk for reactivation, it is crucial that the burden of latent infection be addressed if attempts to control the TB epidemic are to succeed. Despite the centrality of reactivation in Mtb pathogenesis, little is known about the mechanisms which maintain latency and permit the later recrudescence of disease. In particular, bacterial factors at play in re-emergence from dormancy remain elusive. Mtb encodes five genes with homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. Rpf is a 16 kDa secreted protein which stimulates a 1000-fold increase in colony counts when added to "dormant" M. luteus. Mtb Rpfs also stimulate growth of stationary phase M. bovis BCG in vitro, yet the in vivo role of this gene family remains incompletely defined. I observed that rpfs can be deleted individually from the Mtb chromosome without obvious growth defects in vitro or in vivo, during acute infection. I have now found, using a murine TB latency model, that the Mtb Rv1009 (rpf) deletion mutant shows markedly delayed kinetics of reactivation following administration of a nitric oxide synthase inhibitor. To our knowledge this is the first Mtb mutant with a specific in vivo defect in reactivation. Fuller understanding of this phenotype should shed light on mechanisms of mycobacterial dormancy and reactivation, and may offer novel therapeutic targets. This proposal addresses the hypothesis that Rv1009 is a secreted factor which stimulates reactivation of "dormant" bacilli and examines the delayed reactivation phenotype of the Rv1009 mutant from three points of view: characterizing the underlying immune mechanisms using murine models of reactivation, exploring an anaerobic dormancy model as an in vitro correlate, and determining whether multi- Rpf knockouts display delayed reactivation. Relevance: Tuberculosis (TB) causes 2 million deaths per year. M. tuberculosis (Mtb) can remain dormant within infected individuals for decades before reactivating to cause disease. Because a better understanding of the reactivation process is critical to control of TB, this proposal focuses on an Mtb gene family believed to play a role in regulating reactivation from the dormant state. [unreadable] [unreadable] [unreadable]